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Plating efficiency measurements and the experimental control of ageing of adult human skin fibroblasts in vitro
PLATING
EFFICIENCY CONTROL
MEASUREMENTS OF AGEING
AND
OF ADULT
FIBROBLASTS
THE EXPERIMENTAL
HUMAN
SKIN
IN VITRO
D. COUZIN Medical
Research Council, Radiobiology
Unit, Harwell,
Didcot,
Oxon 0x11 ORD, UK
SUMMARY Adult human skin fibroblasts were serially cultured by means of eleven protocols differing in inoculum size, duration of culture between passage and the ability of the medium to support cell division. Each protocol was terminated only when there were too few cells for further subculturing. The fraction of the cells of an inoculum adhering to the growth surface was unaffected by serial subculturing or by differences in protocol. The final cell count at the end of a period of culture and the plating efftciency for the next culture diminished progressively with serial subculturing. Nevertheless, the computed number of cell generations per culture period of those cells which divided was unaffected by serial passaging. The total number of cell doublings accruing during an entire protocol depended only on the diration of the period of culture between successive passages which was characteristic of that protocol. The observations can be accounted for quantitatively by the following assumptions. A cell which loses its ability to divide after a given period of culture nevertheless continues to grow in size during the next period of culture. The increase in volume of cell substance during any such period is the same whether or not a cell divides. The second postulate is that the probability of a cell being able to divide at the start of a period of culture is proportional to the probability that it will not lose this ability by the following period of culture.
MATERIALS AND METHODS The work reported here is an investigation of the effects of various methods of con- Cell origin tinuous serial subculturing on the popula- The cells used for this study were diploid ftbroblasts tion lifespan of adult human diploid fibro- derived from the normal skin of a 15-year-old male. Pieces of skin were taken from the left face and neck blasts. Plating efftciency was used as a sim- during plastic surgery and were immersed in Eagle’s ple and rapid measure of changes occurring MEM. Cultures were generated from small auantities of skin placed in chickembryo extract-chicken plasma in the proliferative capacity of each popula- clots inside 30 ml Falcon T-25 flasks containing 5 ml tion of cells as it progressed through a given culture medium. The routine culture medium was MEM plus 10% human AB serum under a 5% carbon serial subculturing procedure. A new and dioxide atmosphere. All cell cultures were incubated at useful definition of population lifespan is 37°C. The cells were removed from the substrate with 0.1% trypsin for counting and further culturing. The derived to tit the kinetic behaviour of the starting material for the experiments was a large batch cells used. This suggests that simple of cells obtained after two serial subculturings of a number of samples which were then bulked and prechanges in subculturing procedures can in- served under liquid nitrogen. dependently and predictably change both Subculturing the lifespan and the calendar rate of ageing Throughout a given protocol each successive subof cell populations. culture was made in an identical manner. Each sub-
116
D. Couzin
Table 1
Protocol
Expts I
2
3
4”
Subcultured every 3.5 days after inoculation Subcultured as soon as confluency reached Subcultured after 3 days of confluency do. after 7 days do. after I4 days do. after 21 days Subcultured every 3.5 days after inoculation
No. of cells in characteristic inoculum (per flask) b”)
Mean no. of doublings Mean duration of plating of subculture fraction per subculture (days) CT) (ci)
2x lo”
3.5 (fixed)
Estimated total no. of doublings of plating fraction
Total no. of population doublings
3.7kO.2 4.2kO.2 5.5ItO.5
88+5 76+4 94+9
20.8 23.1 l8.S
3.6kO.2 4.3kO.2 6.0+0.2
72+4 65+3 6Ok2
25.8 27.6 28.3
58&5 4224 33f4 34*4
23.7 20.0 14.2 15.5
I 2 3
4x IO”
4 5 6
2x IO” 1x10” 4x IO”
7 8 9 IO
1x10” IX 105 1x105 1x105
(5.1+ 3)+0.4 (5.2+ 7)40.5 (5.3+ l4)?0.8 (6.2+2l)t0.9
4.8+0.4 4.7f0.4 4.7kO.6 4.8kO.5
II
2x105
3.5 (fixed)
3.9kO.2
1X105
4.5kO.4 5.620.4 7.OkO.6
-
fi Serum I1 was used for this experiment.
culture was initiated by a fixed number of cells and these were obtained from the immediately preceding subculture. The fixed number was characteristic for each protocol. The duration of a culture between inoculation with cells and the next passage.was also characteristic for each protocol and may be called its characteristic duration. At the time defined by the protocol the cells in the subculture flasks were treated with trypsin and suspended in medium for counting in three replicate hemocytometers (improved Neubauer). The volume calculated to contain the characteristic inoculum was used to inoculate the next subculture. Usually a second sample of the cells from the same flask was used as a feeder layer for the plating efticiency estimate of the next subculture (see later) and a third sample for an estimation of settling efftciency (see later). Of necessity serial subculturing ceased when the cell count at the end of the characteristic duration was found to be less than that required for the characteristic inoculum of the particular protocol. Throughout, cells were grown in 5 ml of MEM plus serum per Falcon T-25 flask (growth area 2.5 cmZ) and after inoculation of cells each flask was gassed with 5 % CO, and placed in an incubator.
The measurement of the fraction of cells which settle (the settling efficiency) A pilot study indicated that effectively all the cells capable of settling and firmly attaching themselves to the growth surfaces of the flasks had done so within 4 h of inoculation. As a routine procedure therefore
known numbers of cells were placed into 30 ml T-25 Flasks containing MEM+AEI serum and after 4-5 h the flasks were gently shaken, the medium poured off and the number of cells in it counted. The remaining attached cells were removed by trypsin and counted.
The measurement of plating efficiency The plating efficiency of a sample of cells is the fraction of cells each capable of forming a clone and the feeder cell technique of Puck & Marcus [23] was used in its determination. The plating efftciency was estimated for most subcultures of each experimental protocol. For estimating the plating efficiency of the nth subculture of a protocol a second characteristic inoculum (in addition to the characteristic inoculum used for initiating the nth subculture) was taken from the (n- 1)th subculture. This second inoculum was irradiated to 3 krad of 250 kV X-rays (100 rad-min-‘), which was sufficient to prevent the formation of clones in culture, and was then used as the feeder layer in the measurement of the plating efficiency of the nth protocol subculture. One hundred cells (the dependent cells), also taken from the (n - 1)th subculture, were added directly onto the feeder layer prepared as above in a Falcon T-25 flask, the total volume of medium being made up to 5 ml. The flasks were incubated for 14 days and then fixed with 1% aqueous Azur A. Colonies with more than 50 cells derived from the 100 dependent cells. were counted under a dissecting microscope. Plating efficiency was usually measured in duplicate.
Control of cell ageing
G
117
H
I
. .1.. .‘.
\
l \.
,‘,L.. .~..:.--,. * 5 10 15
serial subculture no.; ordinate: platteristic inoculum of 2~ IO5cells/flask. The duration of ing efficiency (as a fraction). each culture was 34 days. In all cases the solid lines are best fits to the model. The decline in plating efftciency with serial subculturing for 11 protocols over 4 experiments. Wp$K[2d- I]+ 1) (A-C) Expt 1: Serum type I was used throughout (variablefmodel) and the duration of each subculture was 31 days. Three p,+,= p,;K@-l)+sK different characteristic inocula were used of (A) 2x 105;(B) 1x 105;(C) 4~ IO4cells/flask. and the broken lines are to the model (D-F) Expt 2: Serum type I was used throughout and each subculture was trypsinized at the first signs of P z Vp,vw[~-~l+I) (constantfmodel) confluency. The same three inocula were used as for ++I p,.K(2”-l)+sK expt 1. (D) 2~ 105;(E) 1x 10s;(F) 4x lo4 cells/flask. where d is the computed number of “plating fraction (G-H) Expt 3: Serum type I was used throughout and each subculture was trypsinized after (G) 3 days doublings”, N is the subculture numberp is plating efor (H) 7 days in confluency. The inoculum of 1x IO5 ficiency, s is the measured settling efficiency and W cells/flask was used in both cases. (or V) and K are the model parameters (together with (I) Expt 4: Serum type II was used with a charac- P,).
Fig. 1. Abscissn:
Serum Serum was decanted from whole “clotted” human AI3 blood, Millipore filtered, and frozen. After several batches had been collected they were pooled and refrozen. Two different pools of serum were employed and differed markedly in their ability to support the cloning of dependent cells. The first pool (serum I) gave a more or less linear dependence of plating efficiency on feeder cell density while the second pooi (serum II) consistently showed no influence of feeder cell density on plating efftciency. Serum I could not support cloning in the absence of feeder cells whereas plating ef-
ficiency under serum II was slightly but significantly greater in the absence of feeder cells.
RESULTS Three experiments were done using serum I with a total of ten different protocols and one experiment using serum II with one protocol (table 1). Expt 1: Protocols 1, 2 and 3. Three dif-
I 18
D. Couzin B
Fig. 2. Abscissa:
serial subculture
no.; ordincdrr: final cell no (X 105)
A
c
ferent characteristic inocula were used with cell numbers in the ratio of 10: 5 : 2. Subculturing was carried out at a fixed time of 33 days after inoculation at which time the cells of protocol I were confluent or nearly so. Comparisons between these protocols allow inferences about the effect of inoculum size on the lifespan of these serially subcultured cells. Expt 2: Protocols 4, 5 and 6. Three different characteristic inocula were used as in expt 1 but on each occasion subculturing was done only when the individual culture had reached confluency. Inevitably this was a subjective criterion throughout. Comparisons with expt 1 provide information about the influence of confluency on population lifespan in culture. Expt 3: Protocols 7, 8, 9 and 10. The characteristic inoculum was the same as for protocols 2 and 5 (i.e. 1x IO5cells/flask) but subcultures were made 3-2 1 days after con-
3
at the end of each subculture. Cell counts at the end of each subculture for I1 protocols of serial subculturing (4 expts). (A) Expt 1: Serum type I was used throughout and subculture durations were fixed at 31 days. Three characteristic inocula were used (i) 2X IO5(O-O); (ii) 1X IO” (A-A); and (iii) 4~ lo4 cells/flask (V-D). (B) Expt 2: Serum type I was used throughout and each subculture was trypsinized at the first signs of confluency. The same three inocula were used as for expt I. (C) Expt 3: Serum type I was used throughout and each subculture was trypsinized after (i) 3 (0-O) or (ii) 7 (A-A) or (iii) 14 (V-V) or (iv) 21 days (O-Cl) in confluency. An inoculum of 1x loj cells/flask was used throughout. (D) Expt 4: Serum type II was used with a characteristic inoculum of 2x IO5cells/flask with subculture
fluency had been reached. Comparisons between these protocols and protocols 2 and 5 give information about the effects of prolonged confluency on population lifespan. In all these experiments, serum I was used. Expt 4: Protocol 11 was the same as protocol 1 but serum II was used. There were four replicates and each serial subculturing was terminated before the end point used for expts 1, 2 and 3 (i.e. before the cell count at the end of the last subculture was less than the characteristic inoculum). Comparison with expts 1, 2 and 3 illustrates the effect of two fundamentally different media on the changes which occur in plating efficiency throughout each serial subculturing. Settling efficiency With some exceptions duplicate measures of the settling efficiency were made at each serial subculturing of each protocol. The
Control of cell ageing
119
before falling as above. The existence of a plateau phase is now considered to be the normal finding. The absence of such a may suggest that they behaviour (phase III From fig. 2B it can be seen that the final cell count appeared to be directly related to inoculum size despite the fact that cells were in confluency at the end of each sub-
serial subculture no.; ordinnte: mean projected area of cells (cm’x 10e5). Changes in mean projected area of cells at confluency, as calculated from final counts. (A) Expt 1: Inoculum of 2~10~ cells/flask only as the smaller inocula did not reach confluency. (B) Expt 2; (C) expt 3; and (D) expt 4: The best fitting curves were drawn by eye. Caption as for fig. 2.
Fig. 3. Abscissa:
average value overall was 0.55+0.02. There were no differences with progressive subculturing or between protocols. Plating efficiency Plating efficiency decreased progressively with progressive serial subculturing in a broadly similar manner in all cases (fig. 1). A comparison of fig. 1A with fig. II, which are of two protocols differing only in the type of serum used, suggests that whether or not a feeder layer was required made little difference to the way plating efficiency changed with number of subcultures. Cell numbers at the end of individual periods of culture The final cell number, at the end of the culture period characteristic of a given protocol also decreased more or less continuously with serial subculturing (fig. 2). This differs from the results of Hayflick [15] who found a plateau phase during which the final cell counts remained more or less constant
For each protocol which was allowed to reach confluency, it was observed that the growth surface of each flask was almost entirely covered by cytoplasm. Direct examination showed two morphological populations: (i) small fibroblast-like cells which were observed to divide frequently and (ii) very large round, highly vacuolate cells which in time-lapse cinemicrography were never observed to divide. With serial subculturing the former population took up progressively less of the growth area at confluency and the latter population progressively more. Culture flasks of the same dimensions (25 cm2 bottom area) were used throughout and, on the assumption that each culture was a monolayer at confluency, the mean projected cell area may be estimated from the cell number at the end of each subculture. Fig. 3 shows that cell area increased continuously with progressive subculturing. From fig. 3A, Z3(protocols 1, 4, 5 and 6) it also appears that cell areas at confluency are inversely related to inoculum size. The mean cell area of the first subcultures of expt 3 (fig. 3C) was about 10m5cm2, the same as in Hayflick’s [16] experiments where lo7 cells could be obtained from one Blake bottle of area 100 cm2. The mean cell area in expt 3 had increased by up to 18 &I
Cdl
Kc, / 16 (1978)
120
D. Couzin
Fin. 4. Abscissa: subculture duration (davs); ordinate: total no. of plating fraction doublings. . The relationship between the total number of “olating fraction doubiings” and the fixed or average &ation of the protocol subcultures. The best fitting exponential regression is also given. Expt 4 is not included.
times by the time subculturing was stopped. However, since multi-layering after prolonged confluency was observed under phase contrast, a more accurate picture is probably obtained from expts 1, 2 and 4 (fig. 3A, B, D, respectively). Here the initial cell area was about 2x 10e5cm* and had increased by the end of serial subculturing by up to 8 times. Such increases in cell size are qualitatively consistent with the results of others [2,4, 251. The number of doublings plating fraction
of the
Let no be inoculum size; p, plating efficiency (as a fraction); n,, cell count at the end of subculture; pno, the fraction of cells which plate (the plating fraction). Also let d be the number of doublings of the plating fraction in the duration of a subculture as defined by the relationships n,=pno2d+(s-p)no
cantly during the series of subcultures in any of the protocols, including those which did not allow the cells to reach confluency. The mean values of d are given in table 1. The constancy of values of d implies a linear relationship between n, and p within a given protocol as was observed. This constancy of d contrasts with the continually declining overall population doublings as demonstrated by the decreasing final cell counts (fig. 2). Inoculation confluency
size and time to reach
As stated previously in nine of the above protocols subcultures were consistently allowed to reach confluency. The time interval from inoculation to the first appearance of confluency was constant within a given protocol. It was shorter for larger inocula, as might be expected, but was affected very little by keeping cells in a state of confluency before initiating a new subculture (expt 3). For protocols involving periods of 3 up to 14 days of confluency, the average growth period in the next culture varied only from 5.1 to 5.3 days (table 1) and the value of 6.220.9 days for protocol [lo] with 2 1 day periods of confluency did not significantly exceed the value of 5.6kO.4 days for subculturing at confluency without delay, as in protocol 5. Thus any lag period induced by confluency must have been quite small. The potential number of plating fraction doublings
If N is used to represent a subculture seor (1) quence number of a protocol and N7 is the total number of subcultures for this proton,=pno(2d- l)+sn, I col then NT A value of d was thus derived for each sub- N=lX d, is the total number of doublings of the culture in which plating efficiency had been plating fractions for the considered protomeasured (fig. 1). It did not vary signifi- col. Exp Cell Ra
116 (1978)
Control of cell ageing
I2 1
every 7 days or so that the subculture durations were increased beyond 3f days the “additional doubling potential” which the cells acquired above the “intrinsic potential” was halved. This relationship was not N=, ” affected by allowing the cells to reach con(where d is the mean value of d). This al- fluency nor apparently by the inoculum lows the value of the total number of doub- size (table 1). Table 1 also gives the total number of lings to be estimated. It is necessary to use the mean 8, since in some subcultures p population doublings (i.e. the total number was not measured and hence d could not be of doublings that the characteristic inocula calculated. The estimated total number of appear to have gone through). It can be plating fraction doublings is given in table 1 seen that for protocols l-6 which do not and plotted against the duration of a period keep the cells in confluency there is a negaof subculture in fig. 4. The 8 protocols in- tive correlation between these doublings volving subculture at or beyond confluency and the plating fraction doublings. In conshow a marked increase in Xd with de- trast, for protocols 7-10 that keep the cells crease in length of period of subculture. in confluency for varying periods a positive Cd in the three protocols of expt 1 with a correlation exists. fixed culture period of 3+ days seems to be roughly constant in the region of 80-90 doublings and in this experiment two of the DISCUSSION protocols (2) and (3), involved subculturing The most common observation of cell before confluency. It was found that the exponential rela- counts during serial subculturing is the existionship tence of the plateau phase first observed by Hayflick [ 151.When cells have been estabXd=aexp(-PT)+y lished in serial culture the number of cells where T is average or fixed subculture dura- at confluency remain unchanged for some tion (days) and CY,p and y are model para- time. Eventually there is a sudden and rapid meters, gave a good fit (P=O.S) to the data decline in cell counts. This is usually aspoints as shown in fig. 4 when a=88*12, sociated with a marked increase in large, p=O. 17f0.04 day-’ and y=32+3. The mini- vacuolated senescent cells [4, 251 as has mum number of doublings (y), achieved been observed here. There is evidence [2, when confluency was prolonged, was there- 191to suggest that these large cells do not fore about 32 when the duration of a period divide. These observations together with of subculture was extended to 19 days or those presented here suggest that: more. (i) at any period during serial culturing a It would seem that the minimum ob- fraction of the cells lose their ability to served doubling potential would not have divide. These newly formed sterile cells been altered much by further extending the grow in volume, become vacuolate and take duration of a period of subculturing. The 32 on the general appearance of senescent doublings (i.e. y) may be considered as cells. being an “intrinsic doubling potential”. For (ii) During the plateau phase an equilibThis does not apply meaningfully to protocol 11 which was prematurely terminated and is therefore excluded from this section. Now
Exp
Cell
Rrs
I16 (1978)
122 D. Couzin rium exists between the rate of cell division and the rate of sterile cell formation. (iii) During the senescent phase this equilibrium breaks down and more sterile cells are produced than are replaced by newly formed dividing cells. It appears from the observations presented here that the increase in projected sterile cell area during the senescent phase balances the reduction in the number of dividing cells per inoculum such that the latter can only produce a fixed number of generations per subculture duration. A simple model to explain these changes quantitatively is now presented. The model takes account of the plateau phase although it was not observed.
vide. These may be further subdivided into those cells which were sterile during the previous subculture and those cells which ceased to divide at this particular inoculation. The former remain unchanged in size and are larger than the cells of the growing population while the latter spend the period in subculture growing in size. (3) The non-viable population, the remaining cells which never adhere to the growth surface and are therefore never counted during the routine cell counts. Letting m be the number of doublings of the growing population required for the whole population to grow from n,, to n2, cells, then n,=gn02m+(s-g)no
A simple explanatory model for constant d and diminishing p during serial subculturing
(2)
and from eq. (1) 2+2”-
l)+ 1.
(3) The cells inoculated into the culture flasks If it is assumed that plating efficiency of constant growth area during a particular subculture of a series may be classified into is a measure of growth fraction such that one of three groups for the purposes of analp=Kg where K is constant ysis: (4) (I ) The growing population, those inoculated cells reponsible for the increase in cell then the observed invariance of d within number. The proportional size of this pop- protocols indicates that m is also a conulation is referred to as the growth frac- stant. Since s was found to be invariant tion, g. The size of the cells of this popu- within a given protocol, then sn, is the conlation remain unchanged during serial sub- stant number of settled cells inoculated durculturing. To be in the growing population ing each passage of the protocol and g/s is a cell need only go through a single division the growth fraction of settled cells. in the duration of a subculture. This conLetting g/s = y trasts with a minimum of 5.6 (i.e. log,50) (5) in 14 days in the presence of irradiated then substituting for g in eq. (2) feeder cells to give a countable clone for the measurement of the plating efficiency. n,=-ysno2m+sno(I-y). (6) Hence growth fraction and plating efficienThe fraction of cells from both settled popcy need not be equal in magnitude. (2) The sterile population, the inoculated ulations which form the next subculture is cells which settle and adhere to the growth surface of the culture flask. but do not di-
Control of cell ageing Consider a protocol where the cells reach confluency at the end of each subculture. At the end of the Nth subculture after the growing population has increased during the subculture from yNsno to ~~~2~2~~ cells after mN doublings, a fraction f,j of the growing population is considered to be transferred to the sterile population during trypsinization. If a cell of the sterile population at confluency occupies, on average, an area of growth surface greater than that of a cell of the growing population by a factor of a! then the area covered by cells at the end of the Nth subculture is
and at the end of the (N+ 1)th subculture can be shown to be
snoxv2[mN+mN+I’.[l-jJ +sn,[ 1-yJ a+sn,y, 2”“f, CZ yh.[2mN- I]+ 1 . The growth areas of all the flasks used were the same and, since all the available area appeared to be covered with the cells at confluency, the above two areas were equal. Since it has been assumed that m is a constant within protocols m,=m,=...=m,=...=m and solving the resultant equation for 2” iY=c~
or (1-yN)I(l -yN-JjV).
The first solution applies for all values off,, and yN (except when yN+fN= 1) and, unlike the second solution, can have a direct biological interpretation. Cells newly transferred to the sterile population increase in
I23
area to the same amount as growth population cells would by dividing to form clones. Now it can be shown that
If 1-fN= Wy, where W is constant
(8)
then YN+l=
wy;. 2” (from eq. (7)) yN(2m- 1>+ 1
(9)
W&K[2”- I]+ 1) p~v+l=p,,~K(2~- l)+sK (substituting eqs (3) (4) and (5) in (8)). This gives a three parameter (W, K and pl) reiterative model for the change of plating efficiency with progressive serial subculturing. The best fit models are given with the plating efficiency data in fig. 1 of the text. A model wheref was kept constant is also given in fig. 1 for comparison. It can be seen that the former but not the latter can account for the continually declining plating efficiency observed for each protocol including ones where confluency is not reached (fig. 1B, C). This suggests that the above assumption (eq. (8)) was a reasonable one to make. Eq. (8) in fact states that the probability of a settled cell being in the growing population at the beginning of any subculture is directly proportional to the probability that it will still be in the growing population by the beginning of the next subculture. The essential nature of this model, which is considered to be the simplest explanation of the data presented, is as follows. When a fixed number of cells are put onto a growth surface of fixed size it may be considered to consist initially of two cell types, dividing cells and sterile cells. The pro-
124
D. Couzin
jetted areas of each are initially the same. Each dividing cell will form a clone which increases in area while each sterile cell will increase in area without division such that the average area of each clone equals the average area of each sterile cell at confluency. Each cell may then be considered at the beginning of the subculture as having a certain area which it will completely occupy, either by division or cell growth, in a fixed time. In all subsequent subcultures the cells which were sterile in the previous subculture will already occupy this area while the newly formed sterile cells and the dividing cells will behave as above. The first point to note is that the area to be occupied by a clone remains unchanged during serial subculturing irrespective of the size of the growing population and the fixed number of doublings per subculture. The second point is that division and cell growth only stops at confluency under the conditions of the experiments reported here. Any initial irregularity of sterile cell size due to cell growth before the protocols were started or to multilayering during prolonged confluency in early subcultures would be quickly rectified by the progressive elimination of these cells. This model explains the observed occurrence of very large cells in culture which have never been observed to divide in time-lapse cinemicrographic studies. Also in accordance with the model are the observations that these cells increased markedly with subculturing. The observed direct relationship between inoculum size and cell numbers at confluency is also expected from this model. It appears from this model that there is a dissociation between DNA synthesis and/ or nuclear division and the synthesis of cytoplasm. During their first subculture as sterile cells the newly formed sterile cells are still capable of manufacturing cyto-
plasm at the same rate as the dividing cells. This hypothesis is supported by the tindings of Macieira-Coelho, Ponten and Philipson [ 181who have shown that relatively few later subculture (phase III) cells synthesize DNA during short periods of pulse labelling while RNA synthesis, although reduced in rate, is maintained in all the cells. From this and his own work Cristofalo [4] suggested that, in older populations, cells may be capable of both protein and RNA synthesis while being unable to carry out DNA synthesis or to accomplish cell division. Cristofalo [4] and Simons [25] also observed that later subcultures contained more large cells than earlier subcultures as was expected from the suggestion of Cristofalo [4]. There is further support from more recent work. Bowman, Meek & Daniel [2] concluded that as cell populations age, increase of cell volume is associated with a loss of ability of the larger cells to divide. Martin et al. [ 191found that cells of human skin fibroblast clones which were, at the most, capable of only a few divisions were larger than cells of more highly proliferative clones. A decrease in the fraction of cells capable of division with age in tissue culture has been found by others [6, 12, 201 and the concept has been widely accepted [9, 11, 221, although it has been questioned by Macieira-Coelho [ 181. The main contention of the model, namely that non-dividing sterile cells which are larger than the dividing cells exist and that these gradually come to dominate the inocula leading eventually to the death of the culture after a finite time, has considerable support. An explanation of the plateau phase The widely observed plateau phase, although not detected here, can be explained
Control of cell ageing using the concepts discussed above. A plateau phase occurs when an equilibrium exists between y andf such that the effective growth fraction at the end of each subculture of a series is reduced to its original size at the beginning of the following subculture. From eq. (7) this occurs when YN’I =YN=Y
i.e. when y= 1-fl( l-2-“).
(10)
125
senescence of a fibroblast population in vitro albeit from chronologically young skin. The possibility cannot be excluded that the rather low settling efficiency observed (55%) is also related to senescence. The repeatability of both the settling efficiency measurements and the senescent behaviour of the cells in widely differeing culture conditions suggest that they were intrinsic properties of the cells at the start of these experiments. This may be related in some way to the physiological status of the donor or to the handling of the cells before these experiments were started.
However, if eq. (8) also acts the equilibrium condition of eq. (10) is unstable. Any slight increase inf will cause a continuous and irreversible reduction of y and hence p. If, however, f is kept independent of y then The use of the plating fraction eq. (10) represents a neutral equilibrium doubling potential condition. Any increase or decrease in f will be associated with a lower or higher Quick and meaningful methods both of plateau level respectively. The model for a determining the lifespan and of controlling constantf is illustrated in fig. 1 and shows the rate of cell ageing in culture are necessary when cells in vitro are used as models that a plateau is quickly achieved. It is suggested that the widely observed for the study of ageing in vivo [5] and also biphasic nature of cell counts during con- when investigating long term selection tinuous serial subculturing is associated mechanisms operating in vitro. Diverse with a change from a more or less fixed f methods of measuring in vitro cell age have during both the growth (phase I) and pla- been considered [I 11. It has been shown [14, 15, 16, 261 that normal cells in culture teau phases to a continuously increasingf have a finite lifetime as do normal cells in during the senescent phase. vivo and this finite lifetime has been considered to be a function of the number of The senescence of the cell population subcultures undergone. Other observations The continually declining cell counts shown [ 13, 211 suggested that the lifespan of the above can only be explained by the absence cells studied was related to the total time in of any equilibrium between the rate of cell culture rather than the number of doublings division and the rate of transfer of cells that had occurred. More recent work [3, 7, from the growing to the sterile population, 8, 10, 241 however, strongly supports the the latter being consistently higher than the former view. The techniques employed former. That a senescent population of cells normally involved various methods of arwas obtained from a young donor does resting cell population growth for varying seem rather surprising. However, the ob- periods. servations reported here do appear to be The usefulness of population doublings concerned with the process of progressive or calendar time as meaningful measures
126
D. Couzin
of ageing is brought into question when it is recognised that the fraction of non-dividing cells increases progressively during serial culturing [6, 12, 201. This is especially so when it is known that different subculturing protocols may affect the increase of the non-dividing fraction to different degrees. This problem was examined by Cristaflo & Sharf [6]. They reported linear relationships between both the duration of culture and, more importantly, the percentage of total population doublings accrued, and the logarithm of percentage unlabelled cells. This method allows estimates to be made of the fraction of life-span completed from a single measurement of the fraction of unlabelled cells present. From these considerations then, perhaps the most immediately significant observation reported here is that, for the range of experimental conditions employed, there is an exponential relationship between the calculated total number of plating fraction doublings and the durations of the subcultures. The total plating fraction doubling potential appears to be determined only by the duration of each subculture. The size of the inoculum, however, affects the number of such doublings per subculture and consequently the number of subcultures available. A control of the total plating fraction doubling potential together with the rate at which this potential is “used up” during serial subculturing appears to be possible for adult human skin fibroblasts. This is valid only for plating fraction doublings and not overall population doublings for the ceMs used here. The results reported here then appear to suggest a useful method for controlling the rate of ageing of cells during serial subculturing, especially at times when no plateau of saturation density (i.e. phase II of Hayflick [ 1.51)is encountered.
I wish to acknowledge the technicalhelpof Mr T. R. L. Biggerand the statistical work of Mr D. J. Papworth. I also wish to thank Dr R. H. Mole for many valuable and critical discussions and Dr D. G. Wiernik of the Churchill Hospital, Oxford for supplying the skin.
REFERENCES I. Absher, P M, Absher, R G & Barnes, W D, Exp cell res 88 (1974) 95. 2. Bowman, R D, Meek, R L & Daniel, C W, Exp cell res 93 (1975) 184. 3. Brunk, U, Ericsson, J L E, PontCn. J & Westermark, B, Exp cell res 79 (1973) I. 4. Cristofalo, V J, Ageing in cell and tissue culture (ed E HoletkovB & V J Cristofalo) p. 83. Plenum Press, New York (1970). 5. Cristofalo, V J, Adv gerontol res 4 (1972) 45. 6. Cristofalo, V J & Sharf, B B, Exp cell res 76 (1973) 419. 7. Daniel, C W & Young, L T T, Exp cell res 65 (1971) 27. 8. Dell’Orco, R T, Mertens, J G & Kruse. P F, Exp cell res 77 (1973) 356. 9. Gelfant, S & Smith, J G, Science 178(1972) 357. 10. Goldstein, S & Singal, D P, Exp cell res 88 (1974) 359. II. Good, P I, Cell tissue kinet 5 (1972) 3 19. 12. Grove, G L & Cristofalo, V J, J cell physiol 90 (1976) 415. 13. Hay, R J, Menzies, R A, Morgan, H P 81Strehler, B L, Exp gerontol 3 (1968) 35. 14. Hayflick, L, Exp cell res 23 (1961) 14. 15. - Ibid 37 (1965) 614. 16. Hayflick, L & Moorhead, P S, Exp cell res 25 (1961) 585. 17. Macieira-Coelho, A, Nature 248 (1974) 42 I. 18. Macieira-Coelho, A, PontCn, J & Philipson, L, Exp cell res 42 (1966) 673. 19. Mat-tin,H M, Sprague,C A, Norwood, T H & Pendergrass, W R, Am j pathol 74 (1974) 137. 20. Merz, G S & Ross, J D, J cell physiol 74 (1969) 219. 21. McHale, J S, Mouton, M L & McHale, J T, Exp eeronto16 (1971) 89. 22. &gel, L E; Natbre 243 (1973) 441. 23. Puck, T T & Marcus, PI, Proc natl acad sci US 41 (1955) 432. 24. Simons, J W I M, Exp cell res 45 (1967) 336. 25. - Ageingin cell and tissue cultureled E HoleCkova & V J Cristofalo) D. 25. Plenum Press. New York (1970). L 26. Swim, H E & Parker, R F, Am j hyg 66 (1957) 235. Received January 18, 1978 Revised version received April 18, 1978 Accepted April 27, 1978
EFFICIENCY CONTROL
MEASUREMENTS OF AGEING
AND
OF ADULT
FIBROBLASTS
THE EXPERIMENTAL
HUMAN
SKIN
IN VITRO
D. COUZIN Medical
Research Council, Radiobiology
Unit, Harwell,
Didcot,
Oxon 0x11 ORD, UK
SUMMARY Adult human skin fibroblasts were serially cultured by means of eleven protocols differing in inoculum size, duration of culture between passage and the ability of the medium to support cell division. Each protocol was terminated only when there were too few cells for further subculturing. The fraction of the cells of an inoculum adhering to the growth surface was unaffected by serial subculturing or by differences in protocol. The final cell count at the end of a period of culture and the plating efftciency for the next culture diminished progressively with serial subculturing. Nevertheless, the computed number of cell generations per culture period of those cells which divided was unaffected by serial passaging. The total number of cell doublings accruing during an entire protocol depended only on the diration of the period of culture between successive passages which was characteristic of that protocol. The observations can be accounted for quantitatively by the following assumptions. A cell which loses its ability to divide after a given period of culture nevertheless continues to grow in size during the next period of culture. The increase in volume of cell substance during any such period is the same whether or not a cell divides. The second postulate is that the probability of a cell being able to divide at the start of a period of culture is proportional to the probability that it will not lose this ability by the following period of culture.
MATERIALS AND METHODS The work reported here is an investigation of the effects of various methods of con- Cell origin tinuous serial subculturing on the popula- The cells used for this study were diploid ftbroblasts tion lifespan of adult human diploid fibro- derived from the normal skin of a 15-year-old male. Pieces of skin were taken from the left face and neck blasts. Plating efftciency was used as a sim- during plastic surgery and were immersed in Eagle’s ple and rapid measure of changes occurring MEM. Cultures were generated from small auantities of skin placed in chickembryo extract-chicken plasma in the proliferative capacity of each popula- clots inside 30 ml Falcon T-25 flasks containing 5 ml tion of cells as it progressed through a given culture medium. The routine culture medium was MEM plus 10% human AB serum under a 5% carbon serial subculturing procedure. A new and dioxide atmosphere. All cell cultures were incubated at useful definition of population lifespan is 37°C. The cells were removed from the substrate with 0.1% trypsin for counting and further culturing. The derived to tit the kinetic behaviour of the starting material for the experiments was a large batch cells used. This suggests that simple of cells obtained after two serial subculturings of a number of samples which were then bulked and prechanges in subculturing procedures can in- served under liquid nitrogen. dependently and predictably change both Subculturing the lifespan and the calendar rate of ageing Throughout a given protocol each successive subof cell populations. culture was made in an identical manner. Each sub-
116
D. Couzin
Table 1
Protocol
Expts I
2
3
4”
Subcultured every 3.5 days after inoculation Subcultured as soon as confluency reached Subcultured after 3 days of confluency do. after 7 days do. after I4 days do. after 21 days Subcultured every 3.5 days after inoculation
No. of cells in characteristic inoculum (per flask) b”)
Mean no. of doublings Mean duration of plating of subculture fraction per subculture (days) CT) (ci)
2x lo”
3.5 (fixed)
Estimated total no. of doublings of plating fraction
Total no. of population doublings
3.7kO.2 4.2kO.2 5.5ItO.5
88+5 76+4 94+9
20.8 23.1 l8.S
3.6kO.2 4.3kO.2 6.0+0.2
72+4 65+3 6Ok2
25.8 27.6 28.3
58&5 4224 33f4 34*4
23.7 20.0 14.2 15.5
I 2 3
4x IO”
4 5 6
2x IO” 1x10” 4x IO”
7 8 9 IO
1x10” IX 105 1x105 1x105
(5.1+ 3)+0.4 (5.2+ 7)40.5 (5.3+ l4)?0.8 (6.2+2l)t0.9
4.8+0.4 4.7f0.4 4.7kO.6 4.8kO.5
II
2x105
3.5 (fixed)
3.9kO.2
1X105
4.5kO.4 5.620.4 7.OkO.6
-
fi Serum I1 was used for this experiment.
culture was initiated by a fixed number of cells and these were obtained from the immediately preceding subculture. The fixed number was characteristic for each protocol. The duration of a culture between inoculation with cells and the next passage.was also characteristic for each protocol and may be called its characteristic duration. At the time defined by the protocol the cells in the subculture flasks were treated with trypsin and suspended in medium for counting in three replicate hemocytometers (improved Neubauer). The volume calculated to contain the characteristic inoculum was used to inoculate the next subculture. Usually a second sample of the cells from the same flask was used as a feeder layer for the plating efticiency estimate of the next subculture (see later) and a third sample for an estimation of settling efftciency (see later). Of necessity serial subculturing ceased when the cell count at the end of the characteristic duration was found to be less than that required for the characteristic inoculum of the particular protocol. Throughout, cells were grown in 5 ml of MEM plus serum per Falcon T-25 flask (growth area 2.5 cmZ) and after inoculation of cells each flask was gassed with 5 % CO, and placed in an incubator.
The measurement of the fraction of cells which settle (the settling efficiency) A pilot study indicated that effectively all the cells capable of settling and firmly attaching themselves to the growth surfaces of the flasks had done so within 4 h of inoculation. As a routine procedure therefore
known numbers of cells were placed into 30 ml T-25 Flasks containing MEM+AEI serum and after 4-5 h the flasks were gently shaken, the medium poured off and the number of cells in it counted. The remaining attached cells were removed by trypsin and counted.
The measurement of plating efficiency The plating efficiency of a sample of cells is the fraction of cells each capable of forming a clone and the feeder cell technique of Puck & Marcus [23] was used in its determination. The plating efftciency was estimated for most subcultures of each experimental protocol. For estimating the plating efficiency of the nth subculture of a protocol a second characteristic inoculum (in addition to the characteristic inoculum used for initiating the nth subculture) was taken from the (n- 1)th subculture. This second inoculum was irradiated to 3 krad of 250 kV X-rays (100 rad-min-‘), which was sufficient to prevent the formation of clones in culture, and was then used as the feeder layer in the measurement of the plating efficiency of the nth protocol subculture. One hundred cells (the dependent cells), also taken from the (n - 1)th subculture, were added directly onto the feeder layer prepared as above in a Falcon T-25 flask, the total volume of medium being made up to 5 ml. The flasks were incubated for 14 days and then fixed with 1% aqueous Azur A. Colonies with more than 50 cells derived from the 100 dependent cells. were counted under a dissecting microscope. Plating efficiency was usually measured in duplicate.
Control of cell ageing
G
117
H
I
. .1.. .‘.
\
l \.
,‘,L.. .~..:.--,. * 5 10 15
serial subculture no.; ordinate: platteristic inoculum of 2~ IO5cells/flask. The duration of ing efficiency (as a fraction). each culture was 34 days. In all cases the solid lines are best fits to the model. The decline in plating efftciency with serial subculturing for 11 protocols over 4 experiments. Wp$K[2d- I]+ 1) (A-C) Expt 1: Serum type I was used throughout (variablefmodel) and the duration of each subculture was 31 days. Three p,+,= p,;K@-l)+sK different characteristic inocula were used of (A) 2x 105;(B) 1x 105;(C) 4~ IO4cells/flask. and the broken lines are to the model (D-F) Expt 2: Serum type I was used throughout and each subculture was trypsinized at the first signs of P z Vp,vw[~-~l+I) (constantfmodel) confluency. The same three inocula were used as for ++I p,.K(2”-l)+sK expt 1. (D) 2~ 105;(E) 1x 10s;(F) 4x lo4 cells/flask. where d is the computed number of “plating fraction (G-H) Expt 3: Serum type I was used throughout and each subculture was trypsinized after (G) 3 days doublings”, N is the subculture numberp is plating efor (H) 7 days in confluency. The inoculum of 1x IO5 ficiency, s is the measured settling efficiency and W cells/flask was used in both cases. (or V) and K are the model parameters (together with (I) Expt 4: Serum type II was used with a charac- P,).
Fig. 1. Abscissn:
Serum Serum was decanted from whole “clotted” human AI3 blood, Millipore filtered, and frozen. After several batches had been collected they were pooled and refrozen. Two different pools of serum were employed and differed markedly in their ability to support the cloning of dependent cells. The first pool (serum I) gave a more or less linear dependence of plating efficiency on feeder cell density while the second pooi (serum II) consistently showed no influence of feeder cell density on plating efftciency. Serum I could not support cloning in the absence of feeder cells whereas plating ef-
ficiency under serum II was slightly but significantly greater in the absence of feeder cells.
RESULTS Three experiments were done using serum I with a total of ten different protocols and one experiment using serum II with one protocol (table 1). Expt 1: Protocols 1, 2 and 3. Three dif-
I 18
D. Couzin B
Fig. 2. Abscissa:
serial subculture
no.; ordincdrr: final cell no (X 105)
A
c
ferent characteristic inocula were used with cell numbers in the ratio of 10: 5 : 2. Subculturing was carried out at a fixed time of 33 days after inoculation at which time the cells of protocol I were confluent or nearly so. Comparisons between these protocols allow inferences about the effect of inoculum size on the lifespan of these serially subcultured cells. Expt 2: Protocols 4, 5 and 6. Three different characteristic inocula were used as in expt 1 but on each occasion subculturing was done only when the individual culture had reached confluency. Inevitably this was a subjective criterion throughout. Comparisons with expt 1 provide information about the influence of confluency on population lifespan in culture. Expt 3: Protocols 7, 8, 9 and 10. The characteristic inoculum was the same as for protocols 2 and 5 (i.e. 1x IO5cells/flask) but subcultures were made 3-2 1 days after con-
3
at the end of each subculture. Cell counts at the end of each subculture for I1 protocols of serial subculturing (4 expts). (A) Expt 1: Serum type I was used throughout and subculture durations were fixed at 31 days. Three characteristic inocula were used (i) 2X IO5(O-O); (ii) 1X IO” (A-A); and (iii) 4~ lo4 cells/flask (V-D). (B) Expt 2: Serum type I was used throughout and each subculture was trypsinized at the first signs of confluency. The same three inocula were used as for expt I. (C) Expt 3: Serum type I was used throughout and each subculture was trypsinized after (i) 3 (0-O) or (ii) 7 (A-A) or (iii) 14 (V-V) or (iv) 21 days (O-Cl) in confluency. An inoculum of 1x loj cells/flask was used throughout. (D) Expt 4: Serum type II was used with a characteristic inoculum of 2x IO5cells/flask with subculture
fluency had been reached. Comparisons between these protocols and protocols 2 and 5 give information about the effects of prolonged confluency on population lifespan. In all these experiments, serum I was used. Expt 4: Protocol 11 was the same as protocol 1 but serum II was used. There were four replicates and each serial subculturing was terminated before the end point used for expts 1, 2 and 3 (i.e. before the cell count at the end of the last subculture was less than the characteristic inoculum). Comparison with expts 1, 2 and 3 illustrates the effect of two fundamentally different media on the changes which occur in plating efficiency throughout each serial subculturing. Settling efficiency With some exceptions duplicate measures of the settling efficiency were made at each serial subculturing of each protocol. The
Control of cell ageing
119
before falling as above. The existence of a plateau phase is now considered to be the normal finding. The absence of such a may suggest that they behaviour (phase III From fig. 2B it can be seen that the final cell count appeared to be directly related to inoculum size despite the fact that cells were in confluency at the end of each sub-
serial subculture no.; ordinnte: mean projected area of cells (cm’x 10e5). Changes in mean projected area of cells at confluency, as calculated from final counts. (A) Expt 1: Inoculum of 2~10~ cells/flask only as the smaller inocula did not reach confluency. (B) Expt 2; (C) expt 3; and (D) expt 4: The best fitting curves were drawn by eye. Caption as for fig. 2.
Fig. 3. Abscissa:
average value overall was 0.55+0.02. There were no differences with progressive subculturing or between protocols. Plating efficiency Plating efficiency decreased progressively with progressive serial subculturing in a broadly similar manner in all cases (fig. 1). A comparison of fig. 1A with fig. II, which are of two protocols differing only in the type of serum used, suggests that whether or not a feeder layer was required made little difference to the way plating efficiency changed with number of subcultures. Cell numbers at the end of individual periods of culture The final cell number, at the end of the culture period characteristic of a given protocol also decreased more or less continuously with serial subculturing (fig. 2). This differs from the results of Hayflick [15] who found a plateau phase during which the final cell counts remained more or less constant
For each protocol which was allowed to reach confluency, it was observed that the growth surface of each flask was almost entirely covered by cytoplasm. Direct examination showed two morphological populations: (i) small fibroblast-like cells which were observed to divide frequently and (ii) very large round, highly vacuolate cells which in time-lapse cinemicrography were never observed to divide. With serial subculturing the former population took up progressively less of the growth area at confluency and the latter population progressively more. Culture flasks of the same dimensions (25 cm2 bottom area) were used throughout and, on the assumption that each culture was a monolayer at confluency, the mean projected cell area may be estimated from the cell number at the end of each subculture. Fig. 3 shows that cell area increased continuously with progressive subculturing. From fig. 3A, Z3(protocols 1, 4, 5 and 6) it also appears that cell areas at confluency are inversely related to inoculum size. The mean cell area of the first subcultures of expt 3 (fig. 3C) was about 10m5cm2, the same as in Hayflick’s [16] experiments where lo7 cells could be obtained from one Blake bottle of area 100 cm2. The mean cell area in expt 3 had increased by up to 18 &I
Cdl
Kc, / 16 (1978)
120
D. Couzin
Fin. 4. Abscissa: subculture duration (davs); ordinate: total no. of plating fraction doublings. . The relationship between the total number of “olating fraction doubiings” and the fixed or average &ation of the protocol subcultures. The best fitting exponential regression is also given. Expt 4 is not included.
times by the time subculturing was stopped. However, since multi-layering after prolonged confluency was observed under phase contrast, a more accurate picture is probably obtained from expts 1, 2 and 4 (fig. 3A, B, D, respectively). Here the initial cell area was about 2x 10e5cm* and had increased by the end of serial subculturing by up to 8 times. Such increases in cell size are qualitatively consistent with the results of others [2,4, 251. The number of doublings plating fraction
of the
Let no be inoculum size; p, plating efficiency (as a fraction); n,, cell count at the end of subculture; pno, the fraction of cells which plate (the plating fraction). Also let d be the number of doublings of the plating fraction in the duration of a subculture as defined by the relationships n,=pno2d+(s-p)no
cantly during the series of subcultures in any of the protocols, including those which did not allow the cells to reach confluency. The mean values of d are given in table 1. The constancy of values of d implies a linear relationship between n, and p within a given protocol as was observed. This constancy of d contrasts with the continually declining overall population doublings as demonstrated by the decreasing final cell counts (fig. 2). Inoculation confluency
size and time to reach
As stated previously in nine of the above protocols subcultures were consistently allowed to reach confluency. The time interval from inoculation to the first appearance of confluency was constant within a given protocol. It was shorter for larger inocula, as might be expected, but was affected very little by keeping cells in a state of confluency before initiating a new subculture (expt 3). For protocols involving periods of 3 up to 14 days of confluency, the average growth period in the next culture varied only from 5.1 to 5.3 days (table 1) and the value of 6.220.9 days for protocol [lo] with 2 1 day periods of confluency did not significantly exceed the value of 5.6kO.4 days for subculturing at confluency without delay, as in protocol 5. Thus any lag period induced by confluency must have been quite small. The potential number of plating fraction doublings
If N is used to represent a subculture seor (1) quence number of a protocol and N7 is the total number of subcultures for this proton,=pno(2d- l)+sn, I col then NT A value of d was thus derived for each sub- N=lX d, is the total number of doublings of the culture in which plating efficiency had been plating fractions for the considered protomeasured (fig. 1). It did not vary signifi- col. Exp Cell Ra
116 (1978)
Control of cell ageing
I2 1
every 7 days or so that the subculture durations were increased beyond 3f days the “additional doubling potential” which the cells acquired above the “intrinsic potential” was halved. This relationship was not N=, ” affected by allowing the cells to reach con(where d is the mean value of d). This al- fluency nor apparently by the inoculum lows the value of the total number of doub- size (table 1). Table 1 also gives the total number of lings to be estimated. It is necessary to use the mean 8, since in some subcultures p population doublings (i.e. the total number was not measured and hence d could not be of doublings that the characteristic inocula calculated. The estimated total number of appear to have gone through). It can be plating fraction doublings is given in table 1 seen that for protocols l-6 which do not and plotted against the duration of a period keep the cells in confluency there is a negaof subculture in fig. 4. The 8 protocols in- tive correlation between these doublings volving subculture at or beyond confluency and the plating fraction doublings. In conshow a marked increase in Xd with de- trast, for protocols 7-10 that keep the cells crease in length of period of subculture. in confluency for varying periods a positive Cd in the three protocols of expt 1 with a correlation exists. fixed culture period of 3+ days seems to be roughly constant in the region of 80-90 doublings and in this experiment two of the DISCUSSION protocols (2) and (3), involved subculturing The most common observation of cell before confluency. It was found that the exponential rela- counts during serial subculturing is the existionship tence of the plateau phase first observed by Hayflick [ 151.When cells have been estabXd=aexp(-PT)+y lished in serial culture the number of cells where T is average or fixed subculture dura- at confluency remain unchanged for some tion (days) and CY,p and y are model para- time. Eventually there is a sudden and rapid meters, gave a good fit (P=O.S) to the data decline in cell counts. This is usually aspoints as shown in fig. 4 when a=88*12, sociated with a marked increase in large, p=O. 17f0.04 day-’ and y=32+3. The mini- vacuolated senescent cells [4, 251 as has mum number of doublings (y), achieved been observed here. There is evidence [2, when confluency was prolonged, was there- 191to suggest that these large cells do not fore about 32 when the duration of a period divide. These observations together with of subculture was extended to 19 days or those presented here suggest that: more. (i) at any period during serial culturing a It would seem that the minimum ob- fraction of the cells lose their ability to served doubling potential would not have divide. These newly formed sterile cells been altered much by further extending the grow in volume, become vacuolate and take duration of a period of subculturing. The 32 on the general appearance of senescent doublings (i.e. y) may be considered as cells. being an “intrinsic doubling potential”. For (ii) During the plateau phase an equilibThis does not apply meaningfully to protocol 11 which was prematurely terminated and is therefore excluded from this section. Now
Exp
Cell
Rrs
I16 (1978)
122 D. Couzin rium exists between the rate of cell division and the rate of sterile cell formation. (iii) During the senescent phase this equilibrium breaks down and more sterile cells are produced than are replaced by newly formed dividing cells. It appears from the observations presented here that the increase in projected sterile cell area during the senescent phase balances the reduction in the number of dividing cells per inoculum such that the latter can only produce a fixed number of generations per subculture duration. A simple model to explain these changes quantitatively is now presented. The model takes account of the plateau phase although it was not observed.
vide. These may be further subdivided into those cells which were sterile during the previous subculture and those cells which ceased to divide at this particular inoculation. The former remain unchanged in size and are larger than the cells of the growing population while the latter spend the period in subculture growing in size. (3) The non-viable population, the remaining cells which never adhere to the growth surface and are therefore never counted during the routine cell counts. Letting m be the number of doublings of the growing population required for the whole population to grow from n,, to n2, cells, then n,=gn02m+(s-g)no
A simple explanatory model for constant d and diminishing p during serial subculturing
(2)
and from eq. (1) 2+2”-
l)+ 1.
(3) The cells inoculated into the culture flasks If it is assumed that plating efficiency of constant growth area during a particular subculture of a series may be classified into is a measure of growth fraction such that one of three groups for the purposes of analp=Kg where K is constant ysis: (4) (I ) The growing population, those inoculated cells reponsible for the increase in cell then the observed invariance of d within number. The proportional size of this pop- protocols indicates that m is also a conulation is referred to as the growth frac- stant. Since s was found to be invariant tion, g. The size of the cells of this popu- within a given protocol, then sn, is the conlation remain unchanged during serial sub- stant number of settled cells inoculated durculturing. To be in the growing population ing each passage of the protocol and g/s is a cell need only go through a single division the growth fraction of settled cells. in the duration of a subculture. This conLetting g/s = y trasts with a minimum of 5.6 (i.e. log,50) (5) in 14 days in the presence of irradiated then substituting for g in eq. (2) feeder cells to give a countable clone for the measurement of the plating efficiency. n,=-ysno2m+sno(I-y). (6) Hence growth fraction and plating efficienThe fraction of cells from both settled popcy need not be equal in magnitude. (2) The sterile population, the inoculated ulations which form the next subculture is cells which settle and adhere to the growth surface of the culture flask. but do not di-
Control of cell ageing Consider a protocol where the cells reach confluency at the end of each subculture. At the end of the Nth subculture after the growing population has increased during the subculture from yNsno to ~~~2~2~~ cells after mN doublings, a fraction f,j of the growing population is considered to be transferred to the sterile population during trypsinization. If a cell of the sterile population at confluency occupies, on average, an area of growth surface greater than that of a cell of the growing population by a factor of a! then the area covered by cells at the end of the Nth subculture is
and at the end of the (N+ 1)th subculture can be shown to be
snoxv2[mN+mN+I’.[l-jJ +sn,[ 1-yJ a+sn,y, 2”“f, CZ yh.[2mN- I]+ 1 . The growth areas of all the flasks used were the same and, since all the available area appeared to be covered with the cells at confluency, the above two areas were equal. Since it has been assumed that m is a constant within protocols m,=m,=...=m,=...=m and solving the resultant equation for 2” iY=c~
or (1-yN)I(l -yN-JjV).
The first solution applies for all values off,, and yN (except when yN+fN= 1) and, unlike the second solution, can have a direct biological interpretation. Cells newly transferred to the sterile population increase in
I23
area to the same amount as growth population cells would by dividing to form clones. Now it can be shown that
If 1-fN= Wy, where W is constant
(8)
then YN+l=
wy;. 2” (from eq. (7)) yN(2m- 1>+ 1
(9)
W&K[2”- I]+ 1) p~v+l=p,,~K(2~- l)+sK (substituting eqs (3) (4) and (5) in (8)). This gives a three parameter (W, K and pl) reiterative model for the change of plating efficiency with progressive serial subculturing. The best fit models are given with the plating efficiency data in fig. 1 of the text. A model wheref was kept constant is also given in fig. 1 for comparison. It can be seen that the former but not the latter can account for the continually declining plating efficiency observed for each protocol including ones where confluency is not reached (fig. 1B, C). This suggests that the above assumption (eq. (8)) was a reasonable one to make. Eq. (8) in fact states that the probability of a settled cell being in the growing population at the beginning of any subculture is directly proportional to the probability that it will still be in the growing population by the beginning of the next subculture. The essential nature of this model, which is considered to be the simplest explanation of the data presented, is as follows. When a fixed number of cells are put onto a growth surface of fixed size it may be considered to consist initially of two cell types, dividing cells and sterile cells. The pro-
124
D. Couzin
jetted areas of each are initially the same. Each dividing cell will form a clone which increases in area while each sterile cell will increase in area without division such that the average area of each clone equals the average area of each sterile cell at confluency. Each cell may then be considered at the beginning of the subculture as having a certain area which it will completely occupy, either by division or cell growth, in a fixed time. In all subsequent subcultures the cells which were sterile in the previous subculture will already occupy this area while the newly formed sterile cells and the dividing cells will behave as above. The first point to note is that the area to be occupied by a clone remains unchanged during serial subculturing irrespective of the size of the growing population and the fixed number of doublings per subculture. The second point is that division and cell growth only stops at confluency under the conditions of the experiments reported here. Any initial irregularity of sterile cell size due to cell growth before the protocols were started or to multilayering during prolonged confluency in early subcultures would be quickly rectified by the progressive elimination of these cells. This model explains the observed occurrence of very large cells in culture which have never been observed to divide in time-lapse cinemicrographic studies. Also in accordance with the model are the observations that these cells increased markedly with subculturing. The observed direct relationship between inoculum size and cell numbers at confluency is also expected from this model. It appears from this model that there is a dissociation between DNA synthesis and/ or nuclear division and the synthesis of cytoplasm. During their first subculture as sterile cells the newly formed sterile cells are still capable of manufacturing cyto-
plasm at the same rate as the dividing cells. This hypothesis is supported by the tindings of Macieira-Coelho, Ponten and Philipson [ 181who have shown that relatively few later subculture (phase III) cells synthesize DNA during short periods of pulse labelling while RNA synthesis, although reduced in rate, is maintained in all the cells. From this and his own work Cristofalo [4] suggested that, in older populations, cells may be capable of both protein and RNA synthesis while being unable to carry out DNA synthesis or to accomplish cell division. Cristofalo [4] and Simons [25] also observed that later subcultures contained more large cells than earlier subcultures as was expected from the suggestion of Cristofalo [4]. There is further support from more recent work. Bowman, Meek & Daniel [2] concluded that as cell populations age, increase of cell volume is associated with a loss of ability of the larger cells to divide. Martin et al. [ 191found that cells of human skin fibroblast clones which were, at the most, capable of only a few divisions were larger than cells of more highly proliferative clones. A decrease in the fraction of cells capable of division with age in tissue culture has been found by others [6, 12, 201 and the concept has been widely accepted [9, 11, 221, although it has been questioned by Macieira-Coelho [ 181. The main contention of the model, namely that non-dividing sterile cells which are larger than the dividing cells exist and that these gradually come to dominate the inocula leading eventually to the death of the culture after a finite time, has considerable support. An explanation of the plateau phase The widely observed plateau phase, although not detected here, can be explained
Control of cell ageing using the concepts discussed above. A plateau phase occurs when an equilibrium exists between y andf such that the effective growth fraction at the end of each subculture of a series is reduced to its original size at the beginning of the following subculture. From eq. (7) this occurs when YN’I =YN=Y
i.e. when y= 1-fl( l-2-“).
(10)
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senescence of a fibroblast population in vitro albeit from chronologically young skin. The possibility cannot be excluded that the rather low settling efficiency observed (55%) is also related to senescence. The repeatability of both the settling efficiency measurements and the senescent behaviour of the cells in widely differeing culture conditions suggest that they were intrinsic properties of the cells at the start of these experiments. This may be related in some way to the physiological status of the donor or to the handling of the cells before these experiments were started.
However, if eq. (8) also acts the equilibrium condition of eq. (10) is unstable. Any slight increase inf will cause a continuous and irreversible reduction of y and hence p. If, however, f is kept independent of y then The use of the plating fraction eq. (10) represents a neutral equilibrium doubling potential condition. Any increase or decrease in f will be associated with a lower or higher Quick and meaningful methods both of plateau level respectively. The model for a determining the lifespan and of controlling constantf is illustrated in fig. 1 and shows the rate of cell ageing in culture are necessary when cells in vitro are used as models that a plateau is quickly achieved. It is suggested that the widely observed for the study of ageing in vivo [5] and also biphasic nature of cell counts during con- when investigating long term selection tinuous serial subculturing is associated mechanisms operating in vitro. Diverse with a change from a more or less fixed f methods of measuring in vitro cell age have during both the growth (phase I) and pla- been considered [I 11. It has been shown [14, 15, 16, 261 that normal cells in culture teau phases to a continuously increasingf have a finite lifetime as do normal cells in during the senescent phase. vivo and this finite lifetime has been considered to be a function of the number of The senescence of the cell population subcultures undergone. Other observations The continually declining cell counts shown [ 13, 211 suggested that the lifespan of the above can only be explained by the absence cells studied was related to the total time in of any equilibrium between the rate of cell culture rather than the number of doublings division and the rate of transfer of cells that had occurred. More recent work [3, 7, from the growing to the sterile population, 8, 10, 241 however, strongly supports the the latter being consistently higher than the former view. The techniques employed former. That a senescent population of cells normally involved various methods of arwas obtained from a young donor does resting cell population growth for varying seem rather surprising. However, the ob- periods. servations reported here do appear to be The usefulness of population doublings concerned with the process of progressive or calendar time as meaningful measures
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of ageing is brought into question when it is recognised that the fraction of non-dividing cells increases progressively during serial culturing [6, 12, 201. This is especially so when it is known that different subculturing protocols may affect the increase of the non-dividing fraction to different degrees. This problem was examined by Cristaflo & Sharf [6]. They reported linear relationships between both the duration of culture and, more importantly, the percentage of total population doublings accrued, and the logarithm of percentage unlabelled cells. This method allows estimates to be made of the fraction of life-span completed from a single measurement of the fraction of unlabelled cells present. From these considerations then, perhaps the most immediately significant observation reported here is that, for the range of experimental conditions employed, there is an exponential relationship between the calculated total number of plating fraction doublings and the durations of the subcultures. The total plating fraction doubling potential appears to be determined only by the duration of each subculture. The size of the inoculum, however, affects the number of such doublings per subculture and consequently the number of subcultures available. A control of the total plating fraction doubling potential together with the rate at which this potential is “used up” during serial subculturing appears to be possible for adult human skin fibroblasts. This is valid only for plating fraction doublings and not overall population doublings for the ceMs used here. The results reported here then appear to suggest a useful method for controlling the rate of ageing of cells during serial subculturing, especially at times when no plateau of saturation density (i.e. phase II of Hayflick [ 1.51)is encountered.
I wish to acknowledge the technicalhelpof Mr T. R. L. Biggerand the statistical work of Mr D. J. Papworth. I also wish to thank Dr R. H. Mole for many valuable and critical discussions and Dr D. G. Wiernik of the Churchill Hospital, Oxford for supplying the skin.
REFERENCES I. Absher, P M, Absher, R G & Barnes, W D, Exp cell res 88 (1974) 95. 2. Bowman, R D, Meek, R L & Daniel, C W, Exp cell res 93 (1975) 184. 3. Brunk, U, Ericsson, J L E, PontCn. J & Westermark, B, Exp cell res 79 (1973) I. 4. Cristofalo, V J, Ageing in cell and tissue culture (ed E HoletkovB & V J Cristofalo) p. 83. Plenum Press, New York (1970). 5. Cristofalo, V J, Adv gerontol res 4 (1972) 45. 6. Cristofalo, V J & Sharf, B B, Exp cell res 76 (1973) 419. 7. Daniel, C W & Young, L T T, Exp cell res 65 (1971) 27. 8. Dell’Orco, R T, Mertens, J G & Kruse. P F, Exp cell res 77 (1973) 356. 9. Gelfant, S & Smith, J G, Science 178(1972) 357. 10. Goldstein, S & Singal, D P, Exp cell res 88 (1974) 359. II. Good, P I, Cell tissue kinet 5 (1972) 3 19. 12. Grove, G L & Cristofalo, V J, J cell physiol 90 (1976) 415. 13. Hay, R J, Menzies, R A, Morgan, H P 81Strehler, B L, Exp gerontol 3 (1968) 35. 14. Hayflick, L, Exp cell res 23 (1961) 14. 15. - Ibid 37 (1965) 614. 16. Hayflick, L & Moorhead, P S, Exp cell res 25 (1961) 585. 17. Macieira-Coelho, A, Nature 248 (1974) 42 I. 18. Macieira-Coelho, A, PontCn, J & Philipson, L, Exp cell res 42 (1966) 673. 19. Mat-tin,H M, Sprague,C A, Norwood, T H & Pendergrass, W R, Am j pathol 74 (1974) 137. 20. Merz, G S & Ross, J D, J cell physiol 74 (1969) 219. 21. McHale, J S, Mouton, M L & McHale, J T, Exp eeronto16 (1971) 89. 22. &gel, L E; Natbre 243 (1973) 441. 23. Puck, T T & Marcus, PI, Proc natl acad sci US 41 (1955) 432. 24. Simons, J W I M, Exp cell res 45 (1967) 336. 25. - Ageingin cell and tissue cultureled E HoleCkova & V J Cristofalo) D. 25. Plenum Press. New York (1970). L 26. Swim, H E & Parker, R F, Am j hyg 66 (1957) 235. Received January 18, 1978 Revised version received April 18, 1978 Accepted April 27, 1978